Regulation Of c-Myc Research Articles

PATH-30. CLINICAL OUTCOMES AND PREDICTIVE BIOMARKERS FOR IDH-WILDTYPE GLIOBLASTOMAS DEVELOPING HYPERMUTATION FOLLOWING TEMOZOLOMIDE TREATMENT

Abstract Current standard of care for IDH-wildtype glioblastoma (GBM) includes maximal safe resection, adjuvant radiation, and chemotherapy with the alkylating agent temozolomide. A subset of gliomas treated with temozolomide develop somatic hypermutation with a predominance of C>T transitions due to the alkylation effects on DNA. However, the frequency of occurrence, clinical ramifications, and predictive biomarkers for developing temozolomide-induced hypermutation (TMZ-HM) in GBM are unknown. Here we performed comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent GBM from 106 patients. Seven patients (7%) developed hypermutation at time of first recurrence, while an additional five patients (5%) developed hypermutation at a subsequent recurrence. Patients who developed TMZ-HM had a significantly longer interval between initial surgery and first recurrence (27.8 vs. 9.9 months, p<0.001). In contrast to IDH-mutant gliomas where TMZ-HM is associated with poor prognosis, those patients with IDH-wildtype GBM who developed TMZ-HM had significantly longer overall survival from both initial surgery (67.9 vs. 20.3 months, p<0.001) and from time of recurrence after development of hypermutation (30.9 vs. 9.5 months, p=0.001). There was no difference in oncogenic alteration frequency in initial GBM that subsequently developed TMZ-HM. However, 4 specific CpG sites out of 12 total interrogated sites in the promoter region of MGMT (a DNA repair enzyme) and 5 specific CpG sites in the promoter region of KCNQ1DN (a negative regulator of c-Myc) were significantly more hypermethylated in those GBM that developed TMZ-HM. We then generated a weighted risk score across these CpG sites that can be used to prospectively identify GBM likely to develop TMZ-HM associated with favorable survival. In summary, patients with GBM that develop TMZ-HM follow a more favorable clinical course, and specific MGMT and KCNQ1DN promoter hypermethylation patterns could serve as a biomarker to identify these patients who may potentially benefit from extended alkylating chemotherapy treatment.

Targeting CREB-binding protein (CBP) abrogates colorectal cancer stemness through epigenetic regulation of C-MYC.

Colorectal cancer (CRC) is a common cancer worldwide with an increasing annual incidence. Cancer stem cells (CSCs) play important roles in the occurrence, development, recurrence, and metastasis of CRC. The molecular mechanism regulating the development of colorectal CSCs remains unclear. The discovery of human induced pluripotent stem cells (hiPSCs) through somatic cell reprogramming has revolutionized the fields of stem cell biology and translational medicine. In the present study, we converted hiPSCs into cancer stem-like cells by culture with conditioned medium (CM) from CRC cells. These transformed cells, termed hiPSC-CSCs, displayed cancer stem-like properties, including a spheroid morphology and the expression of both pluripotency and CSC markers. HiPSC-CSCs showed tumorigenic and metastatic abilities in mouse models. The epithelial-mesenchymal transition phenotype was observed in hiPSC-CSCs, which promoted their migration and angiogenesis. Interestingly, upregulation of C-MYC was observed during the differentiation of hiPSC-CSCs. Mechanistically, CREB binding protein (CBP) bound to the C-MYC promoter, while histone deacetylase 1 and 3 (HDAC1/3) dissociated from the promoter, ultimately leading to an increase in histone acetylation and C-MYC transcriptional activation during the differentiation of hiPSC-CSCs. Pharmacological treatment with a CBP inhibitor or abrogation of CBP expression with a CRISPR/Cas9-based strategy reduced the stemness of hiPSC-CSCs. This study demonstrates for the first time that colorectal CSCs can be generated from hiPSCs. The upregulation of C-MYC via histone acetylation plays a crucial role during the conversion process. Inhibition of CBP is a potential strategy for attenuating the stemness of colorectal CSCs.

Targeted transcriptional downregulation of MYC using epigenomic controllers demonstrates antitumor activity in hepatocellular carcinoma models

Dysregulation of master regulator c-MYC (MYC) plays a central role in hepatocellular carcinoma (HCC) and other cancers but remains an elusive target for therapeutic intervention. MYC expression is epigenetically modulated within naturally occurring DNA loop structures, Insulated Genomic Domains (IGDs). We present a therapeutic approach using an epigenomic controller (EC), a programmable epigenomic mRNA medicine, to precisely modify MYC IGD sub-elements, leading to methylation of MYC regulatory elements and durable downregulation of MYC mRNA transcription. Significant antitumor activity is observed in preclinical models of HCC treated with the MYC-targeted EC, as monotherapy or in combination with tyrosine kinase or immune checkpoint inhibitors. These findings pave the way for clinical development of MYC-targeting epigenomic controllers in HCC patients and provide a framework for programmable epigenomic mRNA therapeutics for cancer and other diseases.

C-Myc alone is enough to reprogram fibroblasts into functional macrophages

BackgroundMacrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases.MethodsThe composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac.ResultsHere we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models.ConclusionsOur findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.

Mechanism of BRD4 Inhibitor-Mediated c-MYC Expression and Regulation of AR Expression to Inhibit Prostate Cancer

Prostate cancer is a major health challenge, probably because of regulatory role of hormones that has not been clearly studied. Our study explored BRD4’s role in the development of prostate cancer. BRD4 expression was detected in tumor tissues by RT-PCR method. Transwell detected cell function after different BRD4 expressions, while Target scan detected the relationship between c-MYC and BRD4, including protein expression between the two luciferase activity and Western-blot. Western-blot detected the protein expression after addition of c-MYC, and further verified the effect of c-MYC expression on AR. We proved that, BRD4 was highly expressed in tumor tissues, and inhibiting BRD4 expression significantly inhibited tumor cell invasion and proliferation. BRD4 targets negative feedback to regulate the expression of c-MYC. Further results showed that, addition of BRD4 activated c-MYC signal transduction and inhibited prostate cancer development. Our study reveals that, BRD4 regulates AR to inhibit prostate cancer by regulating c-MYC. BRD4 is involved in AR-mediated regulation of PCa cells. In addition, inhibiting the expression of BRD4 can inhibit pathological progression of prostate cancer cells.

Vitexicarpin suppresses malignant progression of colorectal cancer through affecting c-Myc ubiquitination by targeting IMPDH2

BackgroundColorectal cancer (CRC) is the second most common cause of cancer-related mortality and is characterised by extensive invasive and metastatic potential. Previous studies have shown that vitexicarpin extracted from the fruits of Vitex rotundifolia can impede tumour progression. However, the molecular mechanisms involved in CRC treatment are still not fully established. PurposeOur study aimed to investigate the anticancer activity, targets, and molecular mechanisms of vitexicarpin in CRC hoping to provide novel therapies for patients with CRC. Study design/MethodsThe impact of vitexicarpin on CRC was assessed through various experiments including MTT, clone formation, EDU, cell cycle, and apoptosis assays, as well as a tumour xenograft model. CETSA, label-free quantitative proteomics, and Biacore were used to identify the vitexicarpin targets. WB, Co-IP, Ubiquitination assay, IF, molecular docking, MST, and cell transfection were used to investigate the mechanism of action of vitexicarpin in CRC cells. Furthermore, we analysed the expression patterns and correlation of target proteins in TCGA and GEPIA datasets and clinical samples. Finally, wound healing, Transwell, tail vein injection model, and tissue section staining were used to demonstrate the antimetastatic effect of vitexicarpin on CRC in vitro and in vivo. ResultsOur findings demonstrated that vitexicarpin exhibits anticancer activity by directly binding to inosine monophosphate dehydrogenase 2 (IMPDH2) and that it promotes c-Myc ubiquitination by disrupting the interaction between IMPDH2 and c-Myc, leading to epithelial-mesenchymal transition (EMT) inhibition. Vitexicarpin hinders the migration and invasion of CRC cells by reversing EMT both in vitro and in vivo. Additionally, these results were validated by the overexpression and knockdown of IMPDH2 in CRC cells. ConclusionThese results demonstrated that vitexicarpin regulates the interaction between IMPDH2 and c-Myc to inhibit CRC proliferation and metastasis both in vitro and in vivo. These discoveries introduce potential molecular targets for CRC treatment and shed light on new mechanisms for c-Myc regulation in tumours.

Suppression of N-Glycosylation of Zinc Finger Protein 471 Affects Proliferation, Invasion, and Docetaxel Sensitivity of Tongue Squamous Cell Carcinoma via Regulation of c-Myc

Zinc finger protein 471 (ZNF471) is a member of the Krüppel-related domain zinc finger protein family, and has recently attracted attention because of its anti-cancer effects. N-glycosylation regulates expression and functions of the protein. This study aimed to investigate the effects of ZNF471 N-glycosylation on the proliferation, invasion, and docetaxel sensitivity of tongue squamous cell carcinoma (TSCC). It analyzed the expression, function, and prognostic significance of ZNF471 in TSCC using bioinformatics techniques such as gene differential expression analysis, univariate Cox regression analysis, functional enrichment analysis, and gene set enrichment analysis. Using site-specific mutagenesis, this study generated three mutant sites for ZNF471 N-glycosylation to determine the effect of N-glycosylation on ZNF471 protein levels and function. Quantitative real-time PCR, Western blot analysis, and immunohistochemistry tests confirmed the down-regulation of ZNF471 expression in TSCC. Low expression of ZNF471 is associated with poor prognosis of patients with TSCC. Overexpression of ZNF471 invitro retarded the proliferation of TSCC cells and suppressed cell invasion and migration ability. Asparagine 358 was identified as a N-glycosylation site of ZNF471. Suppressing N-glycosylation of ZNF471 enhanced the protein stability and promoted the translocation of protein to the cell nucleus. ZNF471 binding to c-Myc gene promoter suppressed oncogene c-Myc expression, thereby playing the anti-cancer effect and enhancing TSCC sensitivity to docetaxel. In all, N-glycosylation of ZNF471 affects the proliferation, invasion, and docetaxel sensitivity of TSCC via regulation of c-Myc.

Abstract 3176: Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer

Abstract Otopetrin 3 (OTOP3) stands out as a pivotal regulator of intracellular pH through its control over proton transportation into cells, affecting fundamental cellular processes. Previous studies have shown that OTOP3 is associated with the prognosis of colorectal cancer (CRC) patients. However, the mechanism underlying OTOP3 regulation remains unclear in cancer. Our investigation reveals OTOP3 as a potent oncogene in CRC, with higher OTOP3 levels correlating with diminished overall survival rates in CRC patients. Comparative analysis between cancerous and normal colon cells highlights a pronounced overexpression of OTOP3 in cancer cells. Suppression of OTOP3 expression significantly curtailed CRC cell proliferation, colony formation, migration, and invasion, concurrently inducing apoptosis and cell cycle arrest, ultimately leading to cancer cell death. Moreover, through CHX, FBS inactivation, and MG132 treatment experiments, it was confirmed that OTOP3 regulates c-Myc expression and stability through ubiquitination and has a critical impact on CRC cell proliferation and growth. Additionally, OTOP3's modulation of HK2, PKM2, and LDHA, underscores its role in impacting cancer cell behavior via glycolytic pathways. In summary, the overexpression of OTOP3 in CRC cells drives cell proliferation and growth through the regulation of c-Myc, impacting crucial cellular processes. Additionally, it promotes these processes by influencing glycolysis. These findings provide valuable insights into potential therapeutic targets for CRC treatment, possibly involving OTOP3 and its associated pathways. Citation Format: Hyeung-Jin Jang, Wona Jee, So-Mi Park, Ye-rin Park, Seok Woo Kim, Ji Hoon Jung. Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3176.

TGFβ signaling pathway in salivary gland tumors

ObjectivePleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFβ signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFβ signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFβ cascade. DesignThirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. ResultsCases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFβ signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. ConclusionsOur study demonstrated the differential expression of TGFβ cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

Elevated expression of HIGD1A drives hepatocellular carcinoma progression by regulating polyamine metabolism through c-Myc–ODC1 nexus

BackgroundHypoxia contributes to cancer progression through various molecular mechanisms and hepatocellular carcinoma (HCC) is one of the most hypoxic malignancies. Hypoxia-inducible gene domain protein-1a (HIGD1A) is typically induced via epigenetic regulation and promotes tumor cell survival during hypoxia. However, the role of HIGD1A in HCC remains unknown.MethodsHIGD1A expression was determined in 24 pairs of human HCC samples and para-tumorous tissues. Loss-of-function experiments were conducted both in vivo and in vitro to explore the role of HIGD1A in HCC proliferation and metastasis.ResultsIncreased HIGD1A expression was found in HCC tissues and cell lines, which was induced by hypoxia or low-glucose condition. Moreover, HIGD1A knockdown in HCC cells arrested the cell cycle at the G2/M phase and promoted hypoxia-induced cell apoptosis, resulting in great inhibition of cell proliferation, migration, and invasion, as well as tumor xenograft formation. Interestingly, these anti-tumor effects were not observed in normal hepatocyte cell line L02. Further, HIGD1A knockdown suppressed the expression of ornithine decarboxylase 1 (ODC1), a rate-limiting enzyme of polyamine metabolism under c-Myc regulation. HIGD1A was found to bind with the c-Myc promoter region, and its knockdown decreased the levels of polyamine metabolites. Consistently, the inhibitory effect on HCC phenotype by HIGD1A silencing could be reversed by overexpression of c-Myc or supplementation of polyamines.ConclusionsOur results demonstrated that HIGD1A activated c-Myc–ODC1 nexus to regulate polyamine synthesis and to promote HCC survival and malignant phenotype, implying that HIGD1A might represent a novel therapeutic target for HCC.

Retraction: SALL4 as an Epithelial-Mesenchymal Transition and Drug Resistance Inducer through the Regulation of c-Myc in Endometrial Cancer.

Retraction: SALL4 as an Epithelial-Mesenchymal Transition and Drug Resistance Inducer through the Regulation of c-Myc in Endometrial Cancer.

The m6A modification-mediated positive feedback between glycolytic lncRNA SLC2A1-DT and c-Myc promotes tumorigenesis of hepatocellular carcinoma.

Glycolysis exerts a key role in the metabolic reprogramming of cancer. Specific long non-coding RNAs (lncRNAs) have been identified to exhibit oncogenic glycolysis regulation. Nevertheless, the precise mechanisms by which glycolysis-related lncRNAs control hepatocellular carcinoma (HCC) are still unknown. We profiled and analyzed glycolysis-associated lncRNA signatures using HCC specimens from The Cancer Genome Atlas (TCGA) dataset. Considerable upregulation of the glycolysis-related lncRNA SLC2A1-DT was noted in HCC tissues; this upregulation was strongly linked with advanced tumor stage and poor prognosis. Cell culture and animal-related studies indicated that knockdown or overexpression of SLC2A1-DT obviously restrained or promoted glycolysis, propagation, and metastasis in HCC cells. Mechanistically, SLC2A1-DT enhanced the interaction of protein between β-catenin and YWHAZ, suppressing the binding between β-catenin and β-TrCP, an E3 ubiquitin ligase. Thereby, SLC2A1-DT impeded the β-TrCP-dependent ubiquitination and β-catenin degradation. The upregulated β-catenin activated the transcription of c-Myc, which then increased the transcription of glycolytic genes including SLC2A1, LDHA, and HK2. Additionally, we revealed that c-Myc transcriptionally induced the expression of methyltransferase 3 (METTL3), which increased N6-methyladenosine (m6A) modification and stability of SLC2A1-DT in a YTHDF1 dependent manner. Collectively, we show that the lncRNA SLC2A1-DT promotes glycolysis and HCC tumorigenesis by a m6A modification-mediated positive feedback mechanism with glycolytic regulator c-Myc and suggested as an innovative treatment option and indicator for HCC.

Novel STAT3 Inhibitor Exerts Anti-breast Cancer Effects Both In vitro and In vivo

Background: Breast cancer is one of the most common malignant tumors. Signal transduction and activators of transcription 3 (STAT3) have been demonstrated to play important roles in breast cancer. However, no direct inhibitor of STAT3 has been approved by the FDA for clinical use. Objective: LL1 is a newly designed STAT3 inhibitor that we identified. In this study, we investigated the cytotoxic effect of LL1 on breast cancer cells and its potential mechanisms. Methods: Colony formation and CCK-8 assay were used to detect the anti-proliferation of LL1. Flow cytometry was used to evaluate mitochondrial membrane potential and apoptosis in breast cancer cells following the treatment of LL1. The expression of proteins was analyzed using western blot, and the invasion and migration of cells were analyzed by wound healing assay and transwell assay. The xenograft model was used to evaluate the anti-cancer effect of LL1 in vivo. Results: LL1 selectively inhibited the expression of p-STAT3, but had no obvious effect on total STAT3. LL1 exhibited great potential in suppressing the proliferation of breast cancer in vitro. Moreover, LL1 induces apoptosis and the decrease of mitochondrial membrane potential in breast cancer cells. LL1 can also inhibit the invasion and migration of breast cancer cells. These cell biology changes may be induced via the regulation of Bcl-2, Bax, cleaved-caspase3, Survivn, Mmp-2, Mmp-9, N-cadherin, E-cadherin, vimentin, c-myc and cyclin D1 by LL1. In addition, LL1 exhibited great antitumor activity in vivo. Conclusion: Our study suggested that LL1 can be considered a promising candidate for the treatment of breast cancer.

The promoting effect and mechanism of MAD2L2 on stemness maintenance and malignant progression in glioma

BackgroundGlioblastoma, the most common primary malignant tumor of the brain, is associated with poor prognosis. Glioblastoma cells exhibit high proliferative and invasive properties, and glioblastoma stem cells (GSCs) have been shown to play a crucial role in the malignant behavior of glioblastoma cells. This study aims to investigate the molecular mechanisms involved in GSCs maintenance and malignant progression.MethodsBioinformatics analysis was performed based on data from public databases to explore the expression profile of Mitotic arrest deficient 2 like 2 (MAD2L2) and its potential function in glioma. The impact of MAD2L2 on glioblastoma cell behaviors was assessed through cell viability assays (CCK8), colony formation assays, 5-Ethynyl-2ʹ-deoxyuridine (EDU) incorporation assays, scratch assays, and transwell migration/invasion assays. The findings from in vitro experiments were further validated in vivo using xenograft tumor model. GSCs were isolated from the U87 and LN229 cell lines through flow cytometry and the stemness characteristics were verified by immunofluorescence staining. The sphere-forming ability of GSCs was examined using the stem cell sphere formation assay. Bioinformatics methods were conducted to identified the potential downstream target genes of MAD2L2, followed by in vitro experimental validation. Furthermore, potential upstream transcription factors that regulate MAD2L2 expression were confirmed through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.ResultsThe MAD2L2 exhibited high expression in glioblastoma samples and showed significant correlation with patient prognosis. In vitro and in vivo experiments confirmed that silencing of MAD2L2 led to decreased proliferation, invasion, and migration capabilities of glioblastoma cells, while decreasing stemness characteristics of glioblastoma stem cells. Conversely, overexpression of MAD2L2 enhanced these malignant behaviors. Further investigation revealed that MYC proto-oncogene (c-MYC) mediated the functional role of MAD2L2 in glioblastoma, which was further validated through a rescue experiment. Moreover, using dual-luciferase reporter gene assays and ChIP assays determined that the upstream transcription factor E2F-1 regulated the expression of MAD2L2.ConclusionOur study elucidated the role of MAD2L2 in maintaining glioblastoma stemness and promoting malignant behaviors through the regulation of c-MYC, suggesting its potential as a therapeutic target.

Metabolic Reprogramming By PRDM16 Drives Cytarabine Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the underlying molecular mechanisms are poorly understood. Here, we show that AML cells with high PRDM16 expression acquire resistance to cytarabine (AraC) through the activation of oxidative phosphorylation (OxPHOS) via regulation of c-MYC. Initially, we established a murine model of AML with high Prdm16 expression. We transduced c-Kit + bone marrow cells from wild-type mice with a retrovirus vector encoding MLL-AF9 (MF9) and the oncogenic short form of Prdm16 (sPrdm16). Using MF9/sPrdm16 cells, we next investigated whether AML cells with high Prdm16 expression exhibited resistance to AraC and/or daunorubicin (DNR), chemotherapeutic agents commonly used for induction chemotherapy. In the cell viability assay, MF9/sPrdm16 cells showed significantly higher IC 50 against AraC, but not against DNR, compared to MF9/control (control) cells, suggesting that high sPrdm16 expression confers relative resistance to AraC on AML cells. Colony-forming assay also confirmed resistant to AraC in MF9/sPrdm16 cells compared to control. Moreover, MF9/sPrdm16 cells were also resistant to AraC in vivo as demonstrated by the shortened survival of mice receiving MF9/sPrdm16 cells. To elucidate the mechanism of AraC resistance, we first examined cell cycle status of MF9/sPrdm16 cells since AraC exerts cytotoxicity in cell cycle dependent manner. To our surprise, MF9/sPrdm16 cells were cycling more rapidly than control cells as demonstrated by decreased G 0 phase and increased S/G 2/M phase. Intriguingly, RNA-sequencing analysis revealed that MF9/sPrdm16 cells displayed a signature of high OxPHOS by gene set enrichment analysis (GSEA). Indeed, oxygen consumption rate, superoxide production from mitochondria, and mitochondrial membrane potential were significantly elevated in MF9/sPrdm16 cells compared to control cells. The inhibition of mitochondrial respiration with metformin, the electron transport chain complex I inhibitor, or tigecycline, the mitochondrial protein synthesis inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells by inducing an energetic shift towards a lower OxPHOS status. These results clearly indicated that sPrdm16 overexpression conferred MF9 cells with AraC resistance byincreasing OxPHOS in mitochondria. Next, we explored the mechanism how sPrdm16 regulates OxPHOS. In addition to elevated OxPHOS signature, RNA-sequencing data also showed strong enrichment of Myc target-signature in MF9/sPrdm16 cells. Western blot analysis showed that sPrdm16 overexpression induced up-regulation of c-Myc and its transcriptional target, Slc1a5, a glutamine transporter. We went on to analyze metabolic changes induced by sPrdm16 overexpression by capillary electrophoresis and mass spectrometry-based metabolome analysis. We found that sPrdm16 induced the accumulation of glutamine, glutamate, α-ketoglutaric acid, succinate, fumarate, and malate in MF9 cells, possibly by activating glutaminolysis via the regulation of c-Myc and Slc1a5 expression, leading to the activation of tricarboxylic acid (TCA) cycle and OxPHOS. We also confirmed the contribution of c-Myc to AraC resistance by overexpressing c-Myc in MF9 cells. As expected, MF9/c-Myc cells mimicked the phenotype of MF9/sPrdm16 cells such as resistance to AraC and high OxPHOS status. Furthermore, 10058-F4, a c-Myc inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells. Next, we asked if the observation in MF9/sPrdm16 cells could be translated into human AML cells. Human AML cell lines, MOLM-13 and THP-1, with sPRDM16 overexpression also showed significantly higher IC 50 against AraC compared to Mock expressed cells. Moreover, RNA-seq data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial showed that AML blasts with high PRDM16 expression demonstrated a gene expression signature associated with “OxPHOS” and “Myc Targets”. In conclusion, our findings demonstrate that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via c-MYC, thereby conferres AraC resistance on AML cells. These results suggest that targeting mitochondrial respiration might be a novel treatment strategy to overcome chemoresistance in AML patients with high PRDM16 expression.

Ischemia-reperfusion injury in human AC16 cardiomyocytes is modulated by AXIN1 depending on c-Myc regulation.

A major consequence of acute myocardial infarction is myocardial ischemia-reperfusion (I/R) injury. Collecting proof demonstrates that AXIN1 assume a basic part in different disease; however, the role of AXIN1 in I/R injury remains to a great extent obscure. The I/R injury model on AC16 cells was constructed. siRNA transfection was used to knockdown AXIN1. The qRT-PCR assays and western blot assays were used to detect the expression level of AXIN1 and other key proteins. CCK-8 assays and cell apoptosis assays were used to detect cell proliferation and cell apoptosis. AXIN1 was significantly overexpressed in an in vitro model of I/R injury. Knockdown of AXIN1 significantly restored the cell proliferation inhibition caused by IR injury, while inhibiting apoptosis and inflammation. Further mechanistic studies revealed that the transcription factor c-Myc could regulate the expression of AXIN1. The effects of I/R injury on AC16 cells after overexpression of c-Myc were reversed by knockdown of AXIN1. Meanwhile, AXIN1 could regulate the SIRT1/p53/Nrf 2 pathway. Our results show an important role for AXIN1 and provide new targets for avoiding and treating I/R injury.

Monocarboxylate transporter 4 protects against myocardial ischemia/reperfusion injury by inducing oxidative phosphorylation/glycolysis interconversion and inhibiting oxidative stress.

Myocardial ischemia/reperfusion (I/R) injury is the primary cause of heart damage in the treatment of myocardial infarction, and the imbalance of the energy metabolism in the pathogenesis of myocardial I/R is one of the main triggers of cardiac dysfunction. Monocarboxylate transporter 4 (MCT4) is a key transporter of lactate, which plays a vital role in cellular metabolism. The present study investigated the role and underlying mechanism of MCT4 in myocardial I/R injury. The results of this study showed that MCT4 was upregulated during oxygen-glucose deprivation (OGD) and restored after reoxygenation in cardiomyocytes HL-1. Interestingly, the overexpression of MCT4 increased cell viability and decreased apoptosis of OGD/R-induced HL-1 cells. Furthermore, MCT4 boosted glucose uptake and lactate levels and promoted protein expression of glycolysis regulator LDHA, while also impeding oxidative phosphorylation (OXPHOS) regulators C-MYC and NDUFB8 in OGD/R-induced HL-1 cells. A reduction in reactive oxygen species and oxidative stress markers malonaldehyde and superoxide dismutase was also observed within the OGD/R stimulated HL-1 cells. Additionally, the in vivo exogenous application of MCT4 restored cardiac function, as demonstrated by the reduced infarct size and decreased myocardial apoptosis in I/R rats. OXPHOS and oxidative stress declined, while glycolysis was activated when the I/R mice were injected with AAV-MCT4. Our findings indicate that MCT4 could exert a cardioprotective effect after myocardial I/R injury by inducing OXPHOS/glycolysis interconversion and inhibiting oxidative stress.

Role of c-Myc in lung cancer: Progress, challenges, and prospects.

Lung cancer remains the leading cause of cancer-related deaths worldwide. Despite the recent advances in cancer therapies, the 5-year survival of non-small cell lung cancer (NSCLC) patients hovers around 20%. Inherent and acquired resistance to therapies (including radiation, chemotherapies, targeted drugs, and combination therapies) has become a significant obstacle in the successful treatment of NSCLC. c-Myc, one of the critical oncoproteins, has been shown to be heavily associated with the malignant cancer phenotype, including rapid proliferation, metastasis, and chemoresistance across multiple cancer types. The c-Myc proto-oncogene is amplified in small cell lung cancers (SCLCs) and overexpressed in over 50% of NSCLCs. c-Myc is known to actively regulate the transcription of cancer stemness genes that are recognized as major contributors to tumor progression and therapeutic resistance; thus, targeting c-Myc either directly or indirectly in mitigation of the cancer stemness phenotype becomes a promising approach for development of a new strategy against drug resistant lung cancers. This review will summarize what is currently known about the mechanisms underlying c-Myc regulation of cancer stemness and its involvement in drug resistance and offer an overview on the current progress and future prospects in therapeutically targeting c-Myc in both SCLC and NSCLC.

A virally encoded GPCR drives glioblastoma through feed-forward activation of the SK1-S1P1 signaling axis.

The G protein-coupled receptor (GPCR) US28 encoded by the human cytomegalovirus (HCMV) is associated with accelerated progression of glioblastomas, aggressive brain tumors with a generally poor prognosis. Here, we showed that US28 increased the malignancy of U251 glioblastoma cells by enhancing signaling mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that stimulates oncogenic pathways in glioblastoma. US28 expression increased the abundance of the key components of the S1P signaling axis, including an enzyme that generates S1P [sphingosine kinase 1 (SK1)], an S1P receptor [S1P receptor 1 (S1P1)], and S1P itself. Enhanced S1P signaling promoted glioblastoma cell proliferation and survival by activating the kinases AKT and CHK1 and the transcriptional regulators cMYC and STAT3 and by increasing the abundance of cancerous inhibitor of PP2A (CIP2A), driving several feed-forward signaling loops. Inhibition of S1P signaling abrogated the proliferative and anti-apoptotic effects of US28. US28 also activated the S1P signaling axis in HCMV-infected cells. This study uncovers central roles for S1P and CIP2A in feed-forward signaling that contributes to the US28-mediated exacerbation of glioblastoma.

Targeting DHODH reveals therapeutic opportunities in ATRA-resistant acute promyelocytic leukemia

Although all-trans retinoic acid (ATRA)-induced differentiation has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological disease, resistance to ATRA in high-risk APL patients remains a clinical challenge. In this paper, we discovered that dihydroorotate dehydrogenase (DHODH) inhibition overcame ATRA resistance. 416, a potent DHODH inhibitor previously obtained in our group, inhibited the occurrence of APL in cells and model mice. Excitingly, 416 effectively overcame ATRA resistance in vitro and in vivo by inducing apoptosis and differentiation. Further mechanistic studies showed that PML/RARα lost the regulation of Bcl-2 and c-Myc in NB4-R1 cells, which probably contributed to ATRA resistance. Notably, 416 maintained its Bcl-2 and c-Myc down-regulation effect in NB4-R1 cells and overcome ATRA resistance by inhibiting DHODH. In conclusion, our study highlights the potential of 416 for APL therapy and overcoming ATRA resistance, supporting the further development of DHODH inhibitors for clinical use in refractory and relapsed APL.